Problems about ligation - (Jun/15/2006 )
friends I'd like your help
Again i have a vector of 4092 bp I make a digstion with EcoRI after I dephosphoriled it, and then its was purified. i obtained 40 ng/ul
From another plasmide I make a digestion with Eco Ri to obtain a frame of 2300 bp I obtained 50 ng/ul.
Now I'd like to obtains a new plasmide with that
but, before to inoculate to the bacteria, i want to be sure, if nos I will have more competent bacteria.
I can obtain a new plasmide
Ligasa T4 from Invitrogen, anda buffer 4X from invitrogen to.
One of you could say me why can not do that.
Guessing that u have a 4092 bp vector and ur insert is 2300bps. u have also dephosphorylated the vector, so now u can go ahead with the ligation. I dont think it should be a problem.
ok i think so, but after to do a electrphoreses i can not see any bands,. why?
I might guess it could be that the spectrophotometer made an error. I am guessing that ur samples have much less DNA as u dont c any bands.
May b start again by digesting higher amount of DNA.
I agree, you should digest more DNA.
Personally, I don't trust in spectophotometric quantitations very much. For ligations or stuff like that I quantify by gel electrophoresis using a quantified ladder (in addition, I can see if my band look o.k., without degradation or mistaken). When I want to be very sure, when I need more exactitude, I do both.